Bio-reactive system and method for voltage controlled metabolism

ABSTRACT

Bio-reactive systems for voltage controlled metabolism are described, that include electrochemical-electrostatic systems having a conventional three electrode cell modified with at least one additional gating electrode. The rate of a metabolic process occurring in at least one organism disposed on a working electrode is controllable by applying a gating voltage VG to the at least one gating electrode. A method for voltage controlled metabolism in a bio-reactive electrostatic cell that includes applying a gating voltage VG to at least one gating electrode is also described. The rate of a metabolic process may he controlled by altering at least one of the magnitude and polarity of the applied gating voltage VG. The method for voltage controlled metabolism may further be used to treat cancer and/or increase the rate of ethanol production by fermentation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/924,072, filed on Jan. 6, 2014, which is hereby incorporated byreference in its entirety.

BACKGROUND

The present application generally relates to anelectrochemical-electrostatic bio-reactive system and method thereof forvoltage controlled metabolism. The electrochemical system generallyrelates to, but is not limited to, amperometry, cyclic voltammetry (CV),linear voltammetry, pulse voltammetry, and the like. The electrostaticsystem consists of two electrodes connected via a voltage source,wherein one electrode is coated with an insulating material so that noelectric current flows in this circuit. In exemplary embodiments, anelectrochemical-electrostatic bio-reactive system applies a voltage toan electrochemical cell in order to control the kinetics of a metabolicreaction taking place in a single organism or organisms in the cell. Inother exemplary embodiments, a method of controlling a metabolicreaction caused by a single organism or organisms includes altering avoltage applied within an electrochemical cell.

Glucose metabolism is the most important and widely studied form ofcarbohydrate metabolism. Glucose metabolism in cells generates energyfor living systems to sustain biological functions. The term “glucosemetabolism” generally refers to the cellular processes that convertglucose to energy for cell utilization. There has been a recent renewedinterest in glucose metabolism due its central role in areas of cellbiology, physiology, medicine and synthetic biology. Effective controlof cellular glucose metabolism has many important future implications,e.g., in developing new cancer therapies and for synthesizing biofuelsfrom organisms.

An emerging theme in cancer research is that metabolic regulation,particularly dealing with glucose metabolism, is intricately linked tocancer formation and progression. The Warburg effect has shown that,compared with normal cells, cancer cells consume much more glucose andmainly process it through aerobic glycolysis. Zhao, Y., Butler, E. B.,and Tan, M., Cell Death and Disease 4, e532; doi:10.1038/cddis.2013.60(2013). Additionally, the theory of quantum metabolism has shown that adifference in metabolic rate exists between normal cells and cancercells using electron transit times (which describes the turnover time ofredox reactions). Davies, P., Demetrius, L. A., and Tuszynski, J. A.,AIP Advances 2, 011101 (2012). This recognized difference in the rate ofglucose consumption between normal cells and cancer cells shows thataltering the rate of glucose metabolism, e.g. lowering the rate ofglucose consumption in cancer cells, may be used in new cancertherapies. Metabolic engineering has also turned towards methods ofcontrolling metabolism for the production of important biofuels. Forexample, cellular metabolic pathways in yeast or bacteria may becontrolled to synthesize compounds or fuels that are difficult orexpensive to produce by other means.

There is presently a dearth of methods for facile control of metabolism.Present methods of controlling metabolism are expensive and/or requirean undue amount of time to conduct. Accordingly, an unmet need existsfor new cancer therapies and accelerated fermentation processes ofmaking biofuels such as ethanol based on the control of metabolicprocesses. Such methods would preferably enable cancer research andprevention methods and/or cheap production of biofuels by controllingthe rate of metabolism in a relatively quick and cost-effective manner.

SUMMARY

The present application provides systems and methods for controlling thekinetics of metabolism by using a voltage source that applies a voltageto an electrically insulated electrode(s) without causing a current inits own circuit. The systems and methods may be used, e.g., in theproduction of alcoholic beverages and ethanol for fuel and/or industrialuse, in the production of other biofuels and biomolecules, in medicalresearch, treatment and imaging, or in food processing applications thatinvolve fermentation. Future applications of the disclosed systems andmethods may include controlling any of the differing forms of metabolismin a single organism or organisms in a container.

More specifically, a diagnostic and treatment method for cancer based onthe Warburg effect may be conducted using the disclosed systems andmethods. Additionally, the disclosed systems and methods can be used tocontrol cellular production of many useful substances including biofuelsand ethanol.

In one aspect, the present invention provides anelectrochemical-electrostatic bio-reactive system for voltage controlledmetabolism according to a first exemplary embodiment comprises a workingelectrode, a reference electrode, and a counter electrode connected in aconventional three electrode electrochemical cell. The referenceelectrode and the counter electrode may be combined in a singleelectrode. The system further includes a gating electrode connected toan external voltage source. The gating electrode may include a piece ofmetal operating as a conductor, where the metal is coated with aninsulator so that the metal is not exposed to the solution in theelectrochemical cell. The metal may be connected through the externalvoltage source to the working electrode. An organism or organisms areplaced in physical contact with the working electrode. The organism ororganisms are operative to cause metabolism of at least one metabolicsubstrate disposed on the organism or organisms. The kinetics of themetabolism is controlled by applying a gating voltage V_(G) via theexternal voltage source between the gating electrode and the workingelectrode, which is in contact with the organism or organisms. A rate ofthe transfer of electrons via/through or within the organism ororganisms may be controllable by applying a gating voltage V_(G) betweenthe gating electrode and the working electrode, which is in contact withthe organism or organisms.

Another aspect of the invention provides anelectrochemical-electrostatic bio-reactive system for voltage controlledmetabolism including a first electrode, a second electrode, and at leastone organism. The at least one organism may be immobilized on or inphysical contact with the first electrode. The at least one organism mayalso be suspended in a solution in the presence of the first electrodeand second electrode without being immobilized on or in physical contactto the electrodes. The second electrode consists of a piece ofmetal/conductor, which is coated with an insulator, the metal/conductorbeing electrically connected via a voltage source to the firstelectrode, which may consist of a piece of metal/conductor, which may becoated with an insulator. The coating insulators prevent the metals ofthe electrodes from being exposed to the solution contained in thesystem.

A method for voltage controlled metabolism in an electrostaticbio-reactive cell according to an exemplary embodiment comprisesdisposing at least one organism on a first electrode or dissolving atleast one organism in a solution in the presence of a first electrodeand a second electrode with either or both electrode coated with aninsulator, contacting the at least one organism with at least onesubstrate present in a solution, applying a gating voltage V_(G) to oneor more second electrodes, which is coated with an insulator, disposedwithin the solution and electrically connected to the first electrodevia the voltage source V_(G), and controlling a rate of a metabolicreaction caused by the at least one organism by selecting at least oneof the magnitude and polarity of the applied gating voltage V_(G).

A method for diagnosing, treating or studying cancer with a voltagecontrolled bio-reactive electrochemical-electrostatic cell or the likecomprises placing in contact with or immobilizing a tumor tissue on aworking electrode, applying a gating voltage V_(G) to one or more gatingelectrodes, which is coated with an insulator and electrically connectedto the working electrode via the voltage source V_(G); and changing therate of tumor tissue formation by applying a gating voltage V_(G).

A method for forming ethanol with a voltage controlledelectrochemical-electrostatic bio-reactive cell comprises immobilizingor making contact with a yeast cell on a working electrode, contactingthe yeast with a glucose or sugar solution, applying a gating voltageV_(G) to one or more gating electrodes, which is coated with aninsulator and disposed within the solution and electrically connectedvia a voltage source to the working electrode; and changing the rate offermentation caused by the yeast cell by applying a gating voltageV_(G).

BRIEF DESCRIPTION OF THE FIGURES

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate some embodiments disclosedherein, and together with the description, serve to explain principlesof the disclosed embodiments.

FIG. 1 is a diagram of an electrochemical-electrostatic system forvoltage controlled metabolism according to a first exemplary embodiment.A conventional three-electrode electrochemical cell is modified with oneor more additional gating electrodes for applying a gating voltage V_(G)to the working electrode, upon which yeast cells are immobilized.V_(cell) is the cell potential. The gating voltage V_(G) can be alteredin order to control the rate of glucose or other sugars metabolism inthe yeast cells. The gating electrodes are electrically connected.

FIG. 2 is a diagram of an electrostatic bio-reactive system for voltagecontrolled metabolism according to a second exemplary embodiment.

FIG. 3(a) is an illustration of a scenario for the transfer of electronsfrom NADH to internal NADH dehydrogenase (NDI) and the subsequenttunneling through the enzyme to ubiquinone (UBQ). It is speculated thatV_(G) causes ions to accumulate around the redox enzyme (oxidoreductase)NDI to induce an electric field that penetrate the enzyme and thereforeto modulate the height of the tunnel barrier between FAD, the activesite of NDI, and UBQ. “ox” and “red” respectively are the oxidized andthe reduced energy levels of FAD and UBQ. This is only an example ofmany interactions between the electron carrier NADH and enzymes inmetabolic pathways.

FIG. 3(b) is an illustration of an altered metabolic pathway in yeastcells which are in contact with the working electrode.

FIG. 4 is a flow chart illustrating a method for voltage controlledmetabolism according to an exemplary embodiment.

FIG. 5 is a set of cyclic voltammograms (CVs) obtained to test thesystem of FIG. 1. A yeast-immobilized graphite working electrode wasexposed to a glucose solution under different conditions to generate CVsdepicted as CVs 1, 2, and 3. CV1 show a control current in just PBSsolution. CV2, obtained in a glucose solution, shows an increased anodiccurrent due to the oxidation of glucose by yeast. CV3 shows a furtherincreased oxidation current caused by application of positive V_(G). CV4was obtained with a bare working electrode in either PBS or a glucosesolution.

FIG. 6(a) is a graph of glucose concentration (mM) v. time (s) obtainedon the system of FIG. 1 under aerobic conditions. Curves 1-4 show adecrease in glucose concentration at progressively faster rates as theamount of V_(G) is increased. Curves 5-7 show a progressively slowerrate of decreasing glucose concentration as the polarity of V_(G) isreversed. Curve 8 is a control trace obtained using a bare electrode.The curves were obtained at room temperature.

FIG. 6(b) is a graph of glucose concentration (mM) v. time (s) obtainedon the system of FIG. 1 under anaerobic conditions. Curves similar tothose illustrated FIG. 5(a) were achieved, however the anaerobic processin FIG. 6(b) shows faster depletion rates for glucose than those in theaerobic process in FIG. 6(a). The curves were obtained at roomtemperature.

FIG. 7 is a scanning electron microscopy (SEM) image of a selected areaof a yeast-immobilized electrode before electrochemical processing whereyeast cells are indicated by the arrow.

FIG. 8 is a graph of Luminescence (×10³ RLU) v. time (s) obtained usinga spectrophotometer with samples processed on the system of FIG. 1 underaerobic conditions. The graph shows the V_(G)-controlled production ofan end product of glucose metabolism, adenosine triphosphate (ATP), withand without V_(G) at different times during a 1-hour period at roomtemperature.

FIG. 9 is a graph of Luminescence (×10³ RLU) v. time (s) obtained usinga spectrophotometer with samples processed on the system of FIG. 1 underanaerobic conditions. The graph shows the V_(G)-controlled production ofan end product of glucose metabolism. ATP, with and without V_(G) atdifferent times over a 1-hour period at room temperature.

FIG. 10 shows graphs of ethanol concentration (% v/v) v. time (h)obtained on the system of FIG. 1 by ebulliometry of electrochemicallyprocessed glucose samples using the system of FIG. 1 under anaerobicconditions. The graphs show the V_(G)-controlled generation of an endproduct of glucose metabolism, ethanol, with and without V_(G) atdifferent times during a 3-hour period at room temperature.

FIG. 11 is a graph of pII v. V_(G) (V) obtained on the system of FIG. 1under anaerobic conditions. The graph shows V_(G)-induced changes in thepH of electrochemically processed 100 mM glucose samples at the end of a1-hour period. The decrease in pH shown is attributed to the formationof an end product of glucose metabolism, CO₂. The curve was obtained atroom temperature.

FIG. 12 is a graph of ethanol concentration (% v/v) v. time (h) obtainedwith glucose samples processed on a modified version of the system ofFIG. 1 including only the working electrode and the gating electrode andthe voltage source that generates V_(G). The graph showsV_(G)-controlled production of ethanol by fermentation over a 3-hourperiod. The curves were obtained at room temperature.

FIG. 13 is a graph of glucose concentration (mM) v. time (h) obtainedwith electrochemically processed glucose samples processed on a modifiedversion of the system of FIG. 1 including only the working electrode andthe gating electrode and the voltage source that generates V_(G). Thegraph shows consumption of glucose concentration at an increased rate asV_(G) is increased over a 3-hour period. The curves were obtained atroom temperature.

FIG. 14 is a graph of ethanol concentration (% v/v) v. time (h) obtainedwith electrochemically processed glucose samples processed on the systemof FIG. 1 according to the first exemplary embodiment under anaerobicconditions. The graph shows an increase and then plateau of ethanolproduction by fermentation as V_(G) is applied over 28 hours. The curveswere obtained at room temperature.

FIG. 15 is a graph of ethanol concentration (% v/v) v. time (h) obtainedwith electrochemically processed glucose samples processed on the systemof FIG. 1 according to the second exemplary embodiment under anaerobicconditions. The graph shows an increase and then plateau of ethanolproduction by fermentation as V_(G) is applied over 24 hours. The curveswere obtained at room temperature.

DETAILED DESCRIPTION

The present application provides a bio-reactive systems for voltagecontrolled metabolism and methods for using the systems. Thebio-reactive system can include an electrostatic-electrochemical systemincluding a conventional three electrode electrochemical cell, or thebio-reactive system can include an electrostatic system including afirst and second electrode(s).

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anorganism” includes a plurality of organisms.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth as used in the specification and claims are to beunderstood as being modified in all instances by the term “about.”Accordingly, unless otherwise indicated, the numerical properties setforth in the following specification and claims are approximations thatmay vary depending on the desired properties sought to be obtained inembodiments of the present invention. Notwithstanding that the numericalranges and parameters setting forth the broad scope of the invention areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical values; however,inherently contain certain errors necessarily resulting from error foundin their respective measurements.

The present application provides a bio-reactive systems for voltagecontrolled metabolism and methods for using the systems. Thebio-reactive system can include an electrostatic-electrochemical systemincluding a conventional three electrode electrochemical cell, or thebio-reactive system can include an electrostatic system including afirst and second electrode.

In one aspect, the present invention provides anelectrochemical-electrostatic bio-reactive system for voltage controlledmetabolism that includes a working electrode, a reference electrode, anda counter electrode connected in a conventional three electrodeelectrochemical cell; at least one gating electrode comprising a metalcoated with an insulator, the gating electrode being electricallyconnected via an external voltage source, that produces a gating voltageV_(G) to the working electrode; and at least one organism disposed onthe working electrode, the at least one organism operative to causemetabolism of at least one metabolic substrate; wherein the kinetics ofthe metabolism is controlled by applying a gating voltage V_(G) via theexternal voltage source between the gating electrode and the workingelectrode, which is in contact with the at least one organism.

With reference to FIG. 1, an electrochemical-electrostatic bio-reactivesystem 100 according to a first exemplary embodiment is illustratedwhich includes a conventional three-electrode electrochemical cellincluding a working electrode 102, a reference electrode 104, and acounter electrode 106. The reference electrode 104 is used to controlthe potential of the working electrode 102 (and the cell current) whilecurrent flows between the working electrode 102 and counter electrode106 through a solution 130 held in container 132. The potentialdifference/voltage between the working electrode and the referenceelectrode is controlled by the voltage source V_(cell) or power supply108.

The term “bio-reactive,” as used herein, refers to a system including abioreactor that supports a biologically active environment. Inparticular, a bioreactor is a vessel in which a chemical process iscarried out which involves organisms or biochemically active substancesderived from such organisms. The term “electrostatic,” as used herein,refers a stationary electric charge or field as opposed to electriccurrents. The term “electrochemical,” as used herein, refers to a systemincluding a three electrode (working, reference and counter)electrochemical cell.

The conventional three-electrode electrochemical cell of FIG. 1 may bemodified with one or more additional gating electrodes 110. The gatingelectrodes 110 are comprised of metal 111 and coated by an insulator112. The metal should be a conductive metal such as silver, copper,gold, platinum, or aluminum. Examples of insulating materials that canbe used to coat the metal 111 include glass, ceramic, and polymers, suchas polyethylene, polystyrene, polypropylene, polyvinyl chloride, nylon,cellulose, and polycarbonate. The one or more gating electrodes 110 arefor applying the gating voltage V_(G) 114 to the working electrode 102.The insulator 112 should be effective to prevent current flow in thegating electrode 110-solution 130-working electrode 102 circuit. Aplurality of gating electrodes 110 may be employed with their metal 111portions electrically connected. The applied gating voltage V_(G) 114may be generated by a voltage source as known to one having ordinaryskill in the art. The one or more gating electrodes 110, the voltagesource V_(G) or power supply 114 and the working electrode 102 form aseparate electrical system relative to the conventional three-electrodecell. Current does not flow in this system because of the insulator 112.In addition, in some embodiments, the reference and counter electrodesare combined into a single electrode.

One or more organisms (e.g., yeast cells) 120 are disposed on theworking electrode 102 which interacts with a metabolic substrate (e.g.,glucose). Organisms suitable for use in the present invention includecells such as eukaryotic or prokaryotic cells. The cells can be singlecells, or they can form part of a colony or tissue. In some embodiments,the organisms are microbial organisms. Examples of suitable organismsinclude yeast and algae.

The term “disposed,” as used herein, refers to organisms that areimmobilized on or in contact with an electrode. For example, an organismcan be intentionally attached to the electrode. Alternately, theorganism can be suspended in a solution and diffuse to the electrode tomake temporary or long term contact to the electrode. Typically, theorganism is be bound to the electrode by physical adsorption, whichinvolves the attractive interaction due to opposite charges. However, insome embodiments, the organism can be chemically linked to the electrodeusing methods known to those skilled in the art.

The bio-reactive system can be used to control the rate of metabolism ofa metabolic substrate by the organism. A metabolic substrate is acompound capable of being metabolized by the organism. The specificmetabolic substrates will therefore vary depending on the particularorganism used in the bio-reactive system. Examples of metabolicsubstrates include fatty acids, oils, and sugars such as glucose,fructose, and galactose. The metabolic substrate may be included in thesolution 130 or in any other manner in which it can be metabolized bythe organism. The bio-reactive system can be used to control variousdifferent types of metabolic reactions. For example, the system can beused to control carbohydrate metabolism, protein metabolism, andphotosynthesis, which involves the metabolism of CO₂.

The cell potential V_(cell) 108 may be such that electrons enter theworking electrode 102 from the yeast cells 120. Movement of electronsbetween the microorganism and the working electrode is one way in whichthe present invention differs from the prior art, in which electrons aresent from an electrode to the microorganism via electron mediators(i.e., charge carriers which are intentionally put into solution) toprovide energy for the microorganism to carry out their metabolism.

The system 100 allows for independent and electrostatic control ofmetabolism in organisms 120 by using the gating voltage V_(G) 114. Forexample, glucose consumption by yeast cells 120 disposed on the workingelectrode 102 can be controlled at will (i.e., increased or decreased)using the gating voltage V_(G) 114. The rate of glucose consumption inthe system 100 has been shown to qualitatively correlate with thevoltage-controlled production of the end products of glucose (C₆H₁₂O₆)metabolism, which are ATP (adenosine triphosphate), ethanol (C₂H₅OH),and carbon dioxide (CO₂). The correlation between consumption of glucoseand formation of end products such as ethanol indicates that system 100is capable of controlling the kinetics of the glucose metabolismreaction in yeast through the applied gating voltage V_(G) 114.

The system 100 can also be viewed from a circuit perspective. In thisaspect, the invention provides an electrochemical-electrostaticbio-reactive system for voltage controlled metabolism according toanother embodiment comprises a first circuit, a second circuit, and atleast one organism. The first circuit comprises a working electrode, areference electrode, and a counter electrode connected in a conventionalthree electrode electrochemical cell. The second circuit comprises agating electrode coated with an insulator and electrically connected tothe working electrode via an external voltage source. No currents flowin the second circuit. The at least one organism is placed in physicalcontact with, or immobilized, on the working electrode. The at least oneorganism is operative to cause metabolism of at least one metabolicsubstrate disposed on the at least one organism. The kinetics of themetabolism is controlled by applying a gating voltage V_(G) via theexternal voltage source between the gating electrode and the workingelectrode, which is in contact with the organism or organisms. A rate ofthe transfer of electrons via/through or within the at least oneorganism may be controllable by applying a gating voltage V_(G) betweenthe gating electrode and the working electrode, which is in contact withthe organism or organisms. V_(G) may also control the transfer ofelectrons between the working electrode and the at least one substratedisposed on the at least one organism via the at least one organism.

Without being bound to any particular theory, V_(G) 114 is the voltageused to electrostatically manipulate the metabolism reaction withoutcausing a current in the gating electrode-working electrode circuit.Depending on factors such as the composition of the working electrode102, the geometry of the working electrode 102, the nature of themetabolic substrate, and the concentration of the metabolic substrate,V_(G) generally has a value of from about −20.0 volt to about 20.0 volt.

According to one embodiment, a positive polarity of V_(G) can be appliedto system 100 to increase the kinetics of a metabolic process occurringin an organism 120. According to another embodiment, a negative polarityof V_(G) may also be applied to system 100 to decrease the kinetics ofthe metabolic process.

An important aspect of metabolism is cellular electron transport. Thefeasibility of controlling electron transfer in biological systems usinga gating voltage has been demonstrated in the reduction of hydrogenperoxide (H₂O₂) at an electrode immobilized with microperoxidase-11,showing controlled kinetics of the bio-catalytic system. Choi, Y. andYau, S. -T. AIP Advances 1, 042175 (2011). Engineered electron transporthas also been achieved in E. coli to produce hydrogen using eliminationof competing reactions, engineering of protein interaction surfaces, andprotein fusion or scaffolding. Agapakis, C. M. et al., Journal ofBiological Engineering 4:3 (2010). Observation on glucose consumptionand the production of metabolic end products using the systems describedin the present application performed with different magnitudes andpolarities of V_(G) provides evidence that the observed controlling ofmetabolism described in the present application is due to controlledcellular electron/charge transport. Song, Y., Wang, J., and Yau, S. -T.Scientific Reports 4, 5429 (2014).

System 100 may control the rate of glucose consumption or depletion inthe presence of yeast (Saccharomyces cerevisiae) by using electrostaticmeans. The gating voltage V_(G) 114 applied to the working electrode 102can be used as a parameter for controlling the kinetics of glucosemetabolism. Advantageously, various embodiments of system 100 provide ameasure of control over metabolism based on a variety of biochemicalreactions. System 100 may control metabolism using a voltage source(V_(G) 114) to control metabolism without dissipating electricalcurrent. Also, system 100 does not require introducing chemicals(mediators) into the solution containing the substrate and matrix wheremetabolism takes place. In some embodiments, the system 100 may becontained within a compartment 132.

The system 100 according to a second exemplary embodiment includes theelectric circuit between the one or more gating electrodes 110, thevoltage source V_(G) 114 and the working electrode 102, which may becoated with an insulator, however forgoes the voltage source V_(cell)108 and the other electrodes normally found in a conventional threeelectrode cell (the reference electrode 104 and counter electrode 106).The electrochemical system is therefore turned off. The at least oneorganism may be suspended in the solution 130 in the presence of bothelectrodes. The at least one organism may be in contact with either orboth electrode(s). The system 100 according to the second exemplaryembodiment is able to control the consumption of a metabolic substrate(e.g., glucose or sugar) and the formation of end products in ametabolic reaction without a current being involved in these processes.

The system 100 can be used to form a variety of different metabolic endproducts, depending on the organism and the substrate being used. Forexample, when the organism is yeast and the substrate is a sugar such asglucose, the system can be used to form ethanol as metabolic endproduct. In some embodiments, the metabolic reaction carried out by thesystem produces at least one end product suitable for use as a biofuel.Examples of biofuels include hydrogen, alkanes such as methane, andalcohols such as ethanol.

FIG. 2 shows the schematic of the actual system, which includes theelectric circuit between the one or more gating (second) electrodes 110and the working (first) electrode 102, however forgoes the otherelectrodes normally found in a conventional three electrode cell (thereference electrode 104 and counter electrode 106). The electrochemicalsystem was turned off.

When under the influence of V_(G), the system 100 of either the firstand second exemplary embodiments changes the consumption of glucosebased on glucose metabolism performed by the yeast cells 120 in contactwith, or immobilized on, the working electrode 102, or the yeast can besuspended in a solution in the container 132. Without being bound to anyparticular theory, V_(G) 114-induced altered metabolism may occur basedon an electrostatic mechanism. Since the one or more gating electrodes110 are electrically insulated, the modification produced by V_(G) 114to the metabolic processes may be of an electrostatic nature. Thedifferent pathways in yeast cells 120 all involve redox reactionscatalysed by redox enzymes. For example, the redox reaction of theNAD⁺/NADH redox couple is catalysed by different dehydrogenases inglycolysis, the Krebs cycle, and the electron transport chain.

Again without being bound by theory, the control of metabolism providedby the systems described herein may be due to the modification of thetunnel barrier for cellular charges; i.e., NAD+ and NADH, by theelectric field induced by V_(G). However, when NAD+ and NADH transferelectrons faster, the organism (e.g., yeast cell) is also beingenergized at a faster rate so that the cells grow faster. Theconsumption of glucose and the production of metabolic end products suchas ethanol and ATP may therefore occur at a faster rate.

It has been previously demonstrated that a gating voltage can be used tocontrol the electron transfer between a redox enzyme and an electrode.See Choi, Y. and Yau, S. -T. AIP Advances 1, 042175 (2011); Choi, Y. andYau, S. -T., Anal. Chem. 81, 7123 (2009). The effect, demonstrated withthe glucose oxidase-glucose system and the microperoxidase-H₂O₂ system,was attributed to the redistribution of charges at thesolution-electrode interface induced by the gating voltage so that anelectric field was set up to modulate the electron tunnel barrier, whichis the protein network between the active site of the enzyme and theelectrode. A similar scenario might also occur in the voltage controlledmetabolism of glucose through applied V_(G) in system 100. The rate ofelectron transfer associated with various redox enzymes/proteins in themetabolic processes can be modulated by the induced field through V_(G).

With reference to FIG. 3(a), a schematic diagram shows the passing ofelectrons from NADII 202 to the yeast's internal NADII dehydrogenase(NDI) 204 situated in the electron transport chain and the subsequenttraversing of the electrons through the FAD center 206 to the ubiquinone(UBQ) 208. FIG. 3(a) further shows the V_(G)-induced charges 209 aroundthe enzyme. The induced charges 209 set up an electric field, whosecomponent opposite to the electron's movement through the tunnel barrierof the enzyme modulates the effective height of the barrier so that theelectron transfer rate can be increased or decreased. See Jackson, J.D., CLASSICAL ELECTRODYNAMICS, 3^(rd) ed., 1998.

Similar modulated electron transfer may also occur with other redoxenzymes/proteins involved in the metabolic process, leading to fasterproduction of end products. Accordingly, organisms other than yeast maybe used in the system and end product formation of these enzymes may becontrolled through applied V_(G).

With reference to FIG. 3(b), a possible description for V_(G)-inducedmetabolism of glucose in yeast is presented. Since the thickness of thecell wall in yeast cells 120 is about 100-200 nm, the extracellularelectron transfer may require a special mechanism. Among the severalspecial mechanisms that have been proposed for extracellular electrontransfer, the direct transfer of electrons via the interaction betweenintracellular electron carriers such as NADH and the trans-plasmamembrane electron transfer (tPMET) system 212, a set of redox enzymesand proteins located in the plasma membrane, may be the most relevantfor understanding the mechanism of V_(G)-induced metabolism of glucose.The tPMET system consists of cytochromes and various redox enzymes suchas NADH oxidase, providing redox activity of the membrane at specificsites.

The transfer of electrons to the working electrode 210 observed in thesystem 100 of FIG. 1 implies possible altered routes for NADHs 202. Withcontinuing reference to FIG. 3(b), possible altered metabolic pathwaysfor the aerobic and anaerobic cases are illustrated. After beinggenerated in glycolysis, instead of diffusing to mitochondria, NADH 202may diffuse to the plasma membrane, where they pass an electron 214 tothe working electrode 210 via the tPMET system 212.

Therefore, in the aerobic case, a fraction of the total NADH 202deviates from the normal pathway by diffusing to the cell wall, whilethe remaining NADH 202 diffuses toward mitochondria to maintain theredox balance needed for sustaining the regular metabolic activity ofthe cell. In the anaerobic case, the amount of NADH 202 that goes to thefermentation process is reduced due to cell wall-bound NADH 202. Thediminished amount of NADH 202 that participates in the normal glucosemetabolisms does not necessarily result in reduced production of endproducts. The amount of the end-products produced in system 100 underthe influence of V_(G) is greater than the normal amount. TheV_(G)-induced increase of metabolic end products may be due to theenhanced electron transfer occurring in glycolysis and Kreb's cycle, andalong the electron transport chain. Therefore, V_(G) controls thekinetics of the metabolic pathways.

Accordingly, the gating voltage V_(G) can be used in system 100 as aparameter for controlling the kinetics of glucose metabolism. Thistechnique of applying V_(G) to alter the rate of metabolic processes mayfind applications in cancer research and diagnosis and in metabolicengineering, where the kinetics of the production of substances can becontrolled using a voltage.

With reference to FIG. 4, a method 5100 for voltage controlledmetabolism is illustrated, starting at S101. At S102, at least oneorganism is immobilized on or in contact with a first electrode in asolution 130. Without being intentionally immobilized or in contact toany electrode, the at least one organism may also be suspended in asolution 130 in the presence of the first electrode and other relevantelectrodes, including a second electrode, which is coated with aninsulator and electrically connected via a voltage source V_(G) to thefirst electrode. According to one embodiment, the at least one organismincludes yeast cells. According to another embodiment, the firstelectrode is a working electrode 102.

The first electrode may or may not be part of a conventional threeelectrode electrochemical cell. According to one embodiment, the firstelectrode is a working electrode 102 contained within a conventionalthree electrode electrochemical cell. According to another embodiment,the electrochemical system of the electrochemical-electrostatic systemis turned off and the first electrode, which may be coated with aninsulating material, is connected via a voltage source V_(G) to thesecond electrode.

At S104, at least one substrate specific for the at least one organismis disposed in a solution 130 so that the at least one substrate isdisposed on the at least one organism. The solution 130 may be heldwithin a container 132.

At S106, a gating voltage V_(G) 114 is applied to one or more secondelectrodes disposed within the solution 130 and electrically connectedvia a voltage source 114 to the working electrode 102. According to oneembodiment, the one or more second electrodes are gating electrodes 110.

At S108, the magnitude of the gating voltage V_(G) is changed in orderto activate the effect of V_(G) on the metabolic process caused by theat least one organism. At S110, the polarity of the gating voltage V_(G)is switched in order to change the normal rate of the metabolic processcaused by the at least one organism.

Steps S108 and S110 may be performed separately or in combination toprovide complex control over the rate of a metabolic process.

This technique may find applications in cancer research and diagnosisand in metabolic engineering, where the kinetics of the production ofsubstances can be controlled using a voltage. In fact, theV_(G)-controlled ethanol production using system 100 shows a possiblerole for external voltage in metabolic engineering.

A method S200 according to another embodiment may be adapted forresearch, the diagnosis or the treatment of cancer based on the Warburgeffect. The same principles described in the method S100 would beapplied to cancer tumor tissues disposed in a solution. V_(G) would beadjusted to change the rate of metabolism of glucose within cancer tumortissues.

A method S300 according to another embodiment may be adapted for theproduction of ethanol by fermentation. The same principles described inmethod S100 would apply, however the at least one organism would includeyeast cells and the substrate is glucose. According to yet anotherembodiment, the at least one organism includes other organisms whichalso produce ethanol by feimentation. The first electrode may or may notbe part of a conventional three electrode electrochemical cell.

In some embodiments, the system of the method can be included in aparticular device or apparatus. For example, in some embodiments, thesystem is included in a biosensor. A biosensor is an analytical device,used for the detection of an analyte, that combines a biologicalcomponent with a physicochemical detector. With regard to the presentinvention, when the system is included in a biosensor, the biologicalcomponent can be the organism of the system, while the physicochemicaldetector is the electrode and other non-organic components of thesystem. The analyte measured by the system will be the variable factor,while other components of the system are held constant. For example, ifthe cells and voltage are constant, the system can be used to measurethe amount of substrate. A biosensor typically includes associatedelectronics or signal processors that are primarily responsible for thedisplay of the results in a user-friendly way, and a container for thesystem and associated electronics and display components.

In another embodiment, the system is included in a biofuel cell. Abiofuel cell is a device that converts the chemical energy frombiological material that serves as fuel into electricity through achemical reaction with oxygen or another oxidizing agent. When thesystem of the invention is included in a biofuel cell, the system of theinvention can be used to generate a metabolic end product that issuitable for use as biofuel, which is then burned in a conventionalbiofuel cell. Alternately, in other embodiments, the electrodes of thesystem serve as the anode and cathode, which together with the othercomponents of the system, generate direct current electricity such thatthe system itself acts as a biofuel cell. In some embodiments, aplurality of biofuel cells can be used which are connected in series toprovide a desired voltage.

The systems and methods described herein can be applied to a variety ofdifferent uses. For example, in one embodiment, the systems can be usedfor the treatment of wastewater. Wastewater treatment includesconversion of undesirable waste products into less harmful materials,often through the action of microorganisms, and the system and methodsof the invention can be used to accelerate this process.

In other embodiments, the system and methods of the invention are usedto generate useful metabolic end products. For example, the inventionincludes methods for performing bioconversion or biotransformationprocesses to produce biofuels. More specifically, the system and/ormethod can be used for performing fermentation using yeast or otherorganisms to produce biofuels, including ethanol. In a related manner,the systems and methods of the invention can be used for performingalgae fermentation of sugar to produce oil and biomass. Alternately, orin addition, the system and methods can be used to create food productsor alcoholic beverages. In further embodiments, specialized cells can beused to make various specialty biotechnology products. For example,B-cells can be used to make antibodies, and proteins such as enzymes orother proteins can be made from a variety of cells, includingrecombinant cells.

In other embodiments, the systems and methods of the invention can beused to diagnose, image, or treat diseases involving substratemetabolism, or cellular electron or charge transport. For example,glucose metabolism plays a significant role in cancer, and therefore thesystems and methods of the invention can be used to diagnose, image, ortreat cancer in a subject. Diseases involving cellular electron orcharge transport include mitochondrial disease in relation to otherdiseases including neurodegenerative conditions (ALS, Alzheimer's,Parkinson's Disease), epilepsy and autism, diseases of thecardiovascular system, liver, and kidney, as well as cancer anddiabetes. Alternately, the systems and methods of the invention can beused to modulate other cells or tissue in otherwise healthy individuals.For example, in some embodiments, the system controls the metabolism ofred blood cells.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES Example 1: An Experimental System for Voltage ControlledMetabolism

As illustrated in FIG. 1, a system for voltage controlled metabolism wasconstructed using the system 100 according to the first exemplaryembodiment. The various steps for construction and operation aredetailed below:

Yeast Preparation and Immobilization on the Working Electrode

Dried baker's yeast (Saccharomyces cerevisiae) was purchased from SigmaAldrich (YSC1) and cultivated for several hours at 30° C. in a solutionof deionized water, glucose and peptone. A yeast-immobilized workingelectrode was prepared by depositing a 0.1 ml drop of the yeast solutionon a 1 mm×1 mm area defined using a mask on a pyrolytic graphite (PG)electrode and incubating the electrode at room temperature for 4 hours.Alternatively, yeast can also diffuse to the electrode withoutimmobilization.

Electrochemical Measurements

A conventional three-electrode electrochemical cell with a volume of 2mL was used to perform electrochemical measurements. Theyeast-immobilized electrodes were used as working electrodes. Acommercial Ag/AgCl (3 M KCl) electrode was used as the referenceelectrode and a platinum wire was used as the counter electrode. A pieceof 0.5 mm-diameter copper wire coated with a thin layer of insulator(enamel) was used as the gating electrode. The wire was bent to formmultiple turns and attached on the working electrode next to theimmobilized yeast. The electrochemical cell was driven by a commercialelectrochemical controller (CH Instruments 66° C.).

Example 2: Cyclic Voltammetry

Cyclic voltammetry of glucose was performed using the system shown inFIG. 1 and as described in Example 1 to show the oxidation of glucose bythe immobilized yeast. The cyclic voltammograms (CVs) in FIG. 5 wereobtained using the yeast-immobilized graphite electrode exposed to aglucose solution under different conditions.

CV1 was obtained in phosphate buffered saline (PBS) whereas CV 2 wasobtained with glucose added to the PBS. CV 1 shows a pair of weak redoxpeaks indicated by the arrows with a formal potential at 50 mV vs. anAg/AgCl reference electrode. Comparing CV2 with CV1 shows increasedanodic current, indicating the oxidation of glucose by the yeast. CV3shows further increase in the oxidation current and enhanced redox peakscaused by the application of a positive V_(G). CV4 was obtained with abare working electrode in PBS and subsequently with glucose added to thePBS.

Example 3: Glucose Concentration Monitoring with Different V_(G) Values

A more direct way of showing the oxidation of glucose is to use thesystem 100 described in FIG. 1 and Example 1 to monitor the change inthe glucose concentration of samples for different values of V_(G).Samples of glucose with a volume of 2 mL in phosphate buffered saline(PBS, 26.8 mM, 2 mL) were processed at room temperature usingyeast-immobilized electrodes at V_(cell)=0.6 V vs. Ag/AgCl for a totaltime of 1200 s. Aliquots of 5 A were taken from the processed samplesevery 300 s to be measured using commercial glucose test strips and aglucose meter, whose measuring range is 20-600 mg/dL (1.11-33.33 mM).The samples were tested at room temperature.

PBS was prepared using de-ionized water (18.2 MΩ-cm). All measurementswere made with PBS at room temperature. BREEZE®2 blood glucose teststrips and a BREEZE®2 blood glucose meter (Bayer Health Care, Mishawaka,Wis.) with a measuring range of 20-600 mg/dL (1.11-33.33 mM) were usedto measure the concentration of glucose in samples.

With reference to FIG. 6(a), the curves show the V_(G)-dependent changein glucose concentration under the aerobic condition. The curves wereobtained with yeast-immobilized electrodes prepared under identicalconditions. Curvet, obtained with V_(G)=0 V, shows a gradual decease inglucose concentration spanning the 1200s period. The effect of apositive V_(G) appears to be causing faster decreases in glucoseconcentration. As V_(G) was increased, the decrease in glucoseconcentration occurred at progressively faster rates as indicated byCurves 2-4. When the polarity of V_(G) was reversed, the rate becameprogressively slower than that of Curve1 as indicated by Curves 5-7.Curve 8 is the control trace obtained using a bare electrode, showing nochange in glucose concentration. The depletion of glucose is presumablyto be due to the metabolism in yeast.

With reference to FIG. 6(b), similar curves were obtained under theanaerobic condition (achieved by purging solution with dry N₂ for 1hour) as shown in FIG. 6(a). Comparing the glucose depletion curves inFIGS. 6(a) and 6(b) shows that the anaerobic process shows fasterdepletion rates than those for the aerobic case. This effect isconsistent with the fact that the rate of glucose consumption is higherunder anaerobic conditions than that observed under aerobic conditionsdue to the fact that the anaerobic process is associated with a lowenergy yield.

Example 4: SEM Image of Immobilized Yeast on Working Electrode

The measurements conducted on system 100 and as described in Example 1were carried out in the lag phase of yeast budding (2-10 hours) to avoidyeast reproduction. With reference to FIG. 7, a scanning electronmicroscopy (SEM) image shows a selected area of an electrode beforecarrying out the electrochemical processing of a glucose sample.

The field emission scanning electron microscope used to image theimmobilized yeast on electrodes was made by Hitachi (FE-SEM 5000). The 3μm×1 μm grain-like structures are yeast cells. SEM images of the samearea after 120 min of electrochemical processing in glucose (image notshown) appear to be identical to the one in FIG. 7, suggesting that thefaster depletion of glucose was not caused by yeast reproduction.

Example 5: Probing End Products of Glucose Metabolism—ATP (Aerobic)

To provide further evidence for the presumed glucose metabolism, the endproducts of the typical metabolic processes were probed. Aerobicmetabolism of glucose is the dismantlement of glucose by glycolysis,Krebs cycle and the electron carriers' traversing the electron-transportchain in the presence of oxygen.

Adenosine triphosphate (ATP) is synthesized during metabolism.Luminescence assay of ATP in yeast cells suspended in glucose samples,which were electrochemically processed using the system 100 described inFIG. 1 as described above, was performed to reveal the ATP produced as afunction of V_(G). The assay of ATP was performed using theBacTiter-Glo™ Microbial Cell Viability Assay kit (Promega, Madison,Wis.). The luminescence was detected using a Victor3 Multilabel PlateCounter (Perkin Elmer) and displayed as relative light units (RLU).

FIG. 8 shows, under the aerobic condition, the amounts of ATP present inthe yeast cells at different times of a 60-min processing period fordifferent V_(G) values. The curves in FIG. 8 show that, as V_(G) becamemore positive, increasing amounts of ATP were produced, whereas theamount of ATP progressively decreased for negative V_(G) values. The ATPproduction is to be compared with the glucose consumption shown in FIG.6(a) to show the correlation between the two V_(G)-dependent processes.This correlation is non-accidental since metabolism of glucose producesATP.

Example 6: Probing End Products of Glucose Metabolism—ATP (Anaerobic)

Under the anaerobic condition, glucose metabolism in yeast proceeds viathe fermentation pathway with the formation of ATP, ethanol and carbondioxide (CO₂) as the end products.

FIG. 9 shows the ATP production under anaerobic conditions. Curve 1,which shows the ATP amount obtained with V_(G)=0, is much less than thecorresponding Curve 1 in the aerobic case (see FIG. 8). This differenceis consistent with the fact that, for yeast, the ATP produced in theaerobic environment is 1.4-5.4 times per mole of glucose more than thatproduced in the anaerobic case. Curve 2 and Curve 3 were obtained with apositive and a negative V_(G), respectively. The effects of the polarityof V_(G) on the production of ATP as indicated by the three curves areconsistent with those for the aerobic case.

Example 7: Probing End Products of Glucose Metabolism—Ethanol(Anaerobic)

Ebulliometry of electrochemically processed glucose samples using thesystem 100 described in FIG. 1 was performed to monitor the generationof ethanol. The curves in FIG. 10 show the generation of ethanol byfermentation occurring in 100 mM glucose samples (30 mL) with andwithout V_(G) at different times during a 3-hour period at roomtemperature. The curves show that positive V_(G) led faster productionof ethanol.

The ethanol concentration of samples (% v/v) was measured using anebulliometer (Dujardin-Salleron, Paris, France) at room temperature. Theworking electrode was a 10 mm v 10 mm carbon cloth.

Example 8: Probing End Products of Glucose Metabolism—CO₂ (Anaerobic)

Measured using a pH meter, the curve in FIG. 11 shows the V_(G)-inducedchanges in the pH of electrochemically processed 100 mM glucose samplesat the end of a 1-hour period. The pH of the samples decreased from 5.9to 5.3 as V_(G) was increased from 0 V to 1 V. It is known that CO₂dissolves slightly in water to form a weak acid, H₂CO₃. The decrease inpH is attributed to the increase in the metabolically produced CO₂content in the samples.

Example 9: Short Time Production of Ethanol By Fermentation—Only Gatingand Working Electrode (No Current Involved in Production)

The system 100 according to the second exemplary embodiment was used toprobe the production of ethanol production (similar to Example 7). Thissystem 100 includes the electric circuit between the one or more gatingelectrodes 110 and the working electrode 102, however forgoes the otherelectrodes normally found in a conventional three electrode cell (thereference electrode 104 and counter electrode 106). The electrochemicalsystem was turned off. The system 100 according to the second exemplaryembodiment is able to control the formation of end products in ametabolic reaction without a current being involved in their production.

With reference to FIG. 12, an applied gating voltage V_(G) of 1Vsignificantly increased the rate of ethanol production by fermentationof 30 mL of a 26.6 mM glucose solution by yeast cells. An applied gatingvoltage V_(G) of 2V produced similar results as 1V. The measurementswere made at room temperature under anaerobic condition. The workingelectrode was a 10 mm×10 mm carbon cloth.

Example 10: Short Time Depletion of Glucose Due to Fermentation—OnlyGating and Working Electrode (No Current Involved in Production)

The system 100 according to the second exemplary embodiment was used tomonitor glucose concentration (similar to Example 3). This system 100includes the electric circuit between the one or more gating electrodes110 and the working electrode 102, however forgoes the other electrodesnormally found in a conventional three electrode cell (the referenceelectrode 104 and counter electrode 106). The system 100 according tothe second exemplary embodiment is able to control the formation of endproducts in a metabolic reaction without a current being involved intheir production.

The glucose concentration measurements in FIG. 13 were made using thesame solutions used in Example 9. The increased rate of production ofethanol in FIG. 12 correlates with the increased rate of glucosedepletion in a 30 mL solution shown in FIG. 13 at varying levels ofV_(G). The measurements were made at room temperature. The workingelectrode was a 10 mm×10 mm carbon cloth. This confirms that ethanol isbeing produced by the fermentation of glucose in yeast cells.

Example 11: Ethanol Production By Fermentation (Anaerobic Metabolism ofGlucose) Over 28 Hours

With reference to FIG. 14, ethanol was produced using the system 100according to the first exemplary embodiment as described in Example 1.This system 100 includes the conventional three-electrode cell and aplurality of gating electrodes as illustrated in FIG. 1. An appliedV_(G) of 3V increased the rate of ethanol formation over the entire 28hour period as compared to the rate with V_(G)=0 V. The increased rateof metabolism under V_(G)=3V plateaued at approximately 11 hours. Thevolume of glucose solution was 250 mL and the starting glucoseconcentration was 1.6 M. The concentration of yeast used was 5 gram/L.The fermentation was carried out at 30° C. under anaerobic condition. A7 cm×13 cm carbon cloth was used as the working electrode. The yeast wascultivated as described in Example 1. Alternatively, yeast can be mixedwith water and glucose without cultivation for immediate fermentationprocess and similar fermentation results were obtained.

Example 12: Ethanol Production By Fermentation (Anaerobic Metabolism ofGlucose) Over 24 Hours Using a Two-Electrode System

With reference to FIG. 15, ethanol was produced using the system 100according to the second exemplary embodiment (See FIG. 2). As describedin Example 1, a piece of 0.5 mm-diameter copper wire coated with a thinlayer of insulator (enamel) was used as the gating (second) electrode.This system is able to control the formation of ethanol by fermentationof glucose without a current being involved in its production.

An applied V_(G) of 20 V increased the rate of ethanol formation overthe entire 24 hour period as compared to the rate with V_(G)=0 V. Theincreased rate of fermentation under V_(G)=20 V plateaued atapproximately 14 hours. The volume of glucose solution was 250 mL andthe starting glucose concentration was 200 g/L. The concentration ofyeast used was 12 gram/L. The fermentation was carried out at 30° C.under anaerobic condition. A 7 cm×13 cm carbon cloth was used as theworking electrode and a copper wire coated with enamel was used as thegating electrode. Dry yeast was mixed with water and glucose and themixture was used immediately for the fermentation process withoutcultivation. Alternatively, dry yeast can be cultivated first asdescribed in Example 1. During the fermentation process, yeast wassuspended in the mixture in the presence of the gating electrodes andworking electrode. No attempt was made to immobilize yeast on theelectrodes.

The complete disclosure of all patents, patent applications, andpublications, and electronically available materials cited herein areincorporated by reference. The foregoing detailed description andexamples have been given for clarity of understanding only. Nounnecessary limitations are to be understood therefrom. In particular,while theories may be presented describing operation of the invention,the inventors are not bound by theories described herein. The inventionis not limited to the exact details shown and described, for variationsobvious to one skilled in the art will be included within the inventiondefined by the claims.

1. An electrostatic bio-reactive system for voltage controlledmetabolism, comprising: a first electrode, wherein the first electrodecomprises a metal, which may be coated with an insulator; a secondelectrode electrically connected through an external voltage sourceproviding V_(G) to the first electrode, wherein the second electrodecomprises a metal coated with an insulator; and at least one organismthat is disposed on the first electrode and/or the second electrode, oris suspended in a solution in the presence of the first electrode andthe second electrode, the at least one organism operative to causemetabolism of a metabolic substrate; and the metabolic substrate;wherein the operation of the system does not involve electric currents;wherein the produced voltage V_(G) controls the kinetics of a metabolicreaction performed by the at least one organism on the metabolicsubstrate.
 2. The system of claim 1, wherein the organism is yeast. 3.The system of claim 1, wherein the organism is algae.
 4. The system ofclaim 1, wherein the metabolic substrate is a sugar.
 5. The system ofclaim 4, wherein the metabolic substrate is glucose.
 6. The system ofclaim 1, wherein the second electrode comprises further a plurality ofelectrically connected electrodes.
 7. The system of claim 1, wherein thesystem is included in a biosensor.
 8. The system of claim 1, wherein thesystem is included in a biofuel cell. 9-34. (canceled)